Injectable microtissue systems, devices, and methods

Abstract

Spheroid microtissues that can mimic native tissue-like structure and function, spheroid production methods that are high-throughput, suitable for efficient production, maintainable over long-term culture, and/or offer repeatable control over size distribution. Spheroids that have blood vessels, including spheroids with functional, blood-perfused vascular networks upon injection in vivo. Dissolvable hydrogel microwell arrays for high throughput parallel formation of spheroids in a single pipetting step and easy retrieval for downstream applications. A method to produce prevascularized microtissues in sufficient numbers to form a macrotissue in vivo for therapeutic purposes. This method is based on sacrificial release of dissolvable microwell templates, a novel and scalable strategy which enables gentle harvesting of microtissues with control over size and composition. The method forms microtissues containing endothelial cells and mesenchymal stem cells, which are co-cultured under dynamic conditions and self-organize into blood-vessel units.

The invention claimed is: 
     
       1. An injectable microtissue comprising:
 a plurality of spheroids, each comprising a plurality of cells of a first cell type and a plurality of cells of a second cell type, the first cell type being endothelial cells that are concentrated in an interior core region of each spheroid, wherein the second cell type is one of mesenchymal stem cells, smooth muscle cells, and dermal fibroblasts; 
 the plurality of spheroids being free of vascular interconnections therebetween and suspended in an injectable medicament, and 
 wherein the injectable microtissue is free of brown adipose tissue, 
 wherein the injectable microtissue is suitable for therapeutic injection in a human patient, 
 wherein a majority of the spheroids have diameters less than 200 μm, 
 wherein the endothelial cells concentrated in an interior region define a distinct structure with discernable boundaries and consisting essentially of said endothelial cells, and 
 wherein the endothelial cells are vascular endothelial cells and the distinct structure is a blood vessel precursor. 
 
     
     
       2. The microtissue of  claim 1 , wherein the plurality of cells of the second type form a shell that can withstand the shear strain and stress of injection through a 27 ga (0.27 mm) or 30 ga (0.159 mm) cannula at rates of 0.1 to 2 mL/min. 
     
     
       3. An injectable microtissue comprising:
 a plurality of spheroids, each comprising a plurality of cells of a first cell type and a plurality of cells of a second cell type, the first cell type being endothelial cells that are concentrated in an interior core region of each spheroid; 
 the plurality of spheroids being free of vascular interconnections therebetween and suspended in an injectable medicament, 
 wherein the injectable microtissue is free of brown adipose tissue, 
 wherein the injectable microtissue is suitable for therapeutic injection in a human patient, 
 wherein a majority of the spheroids have diameters less than 200 μm, and wherein the second cell type is mesenchymal stem cells. 
 
     
     
       4. A method of treating a patient, the method comprising:
 injecting an injectable microtissue into a patient such that a new vascular network is formed therein with the endothelial cell core regions of the spheroids as nodes, 
 wherein the injectable microtissue comprises: 
 a plurality of spheroids, each comprising a plurality of cells of a first cell type and a plurality of cells of a second cell type, the first cell type being endothelial cells that are concentrated in an interior core region of each spheroid, wherein the second cell type is one of mesenchymal stem cells, smooth muscle cells, and dermal fibroblasts; 
 the plurality of spheroids being free of vascular interconnections therebetween and suspended in an injectable medicament, 
 wherein the injectable microtissue is free of brown adipose tissue, 
 wherein the injectable microtissue is suitable for therapeutic injection in a human patient, 
 wherein a majority of the spheroids have diameters less than 200 μm, and 
 wherein the endothelial cells concentrated in an interior region define a distinct structure with discernable boundaries and consisting of said endothelial cells, the endothelial cells are vascular endothelial cells, and the distinct structure is a blood vessel precursor. 
 
     
     
       5. The method of  claim 4 , wherein the injecting is effective to treat a condition of ischemia. 
     
     
       6. The method of  claim 4 , wherein the injecting is performed at multiple locations spaced apart along a limb or other anatomical feature of the patient. 
     
     
       7. The microtissue of  claim 1 , wherein the injectable microtissue is free of growth factors. 
     
     
       8. A method of treating a patient, the method comprising:
 injecting an injectable microtissue into a patient such that a new vascular network is formed therein with the endothelial cell core regions of the spheroids as nodes, 
 wherein the injectable microtissue comprises: 
 a plurality of spheroids, each comprising a plurality of cells of a first cell type and a plurality of cells of a second cell type, the first cell type being endothelial cells that are concentrated in an interior core region of each spheroid; 
 the plurality of spheroids being free of vascular interconnections therebetween and suspended in an injectable medicament, 
 wherein the injectable microtissue is free of brown adipose tissue, 
 wherein the injectable microtissue is suitable for therapeutic injection in a human patient, 
 wherein a majority of the spheroids have diameters less than 200 μm, and 
 wherein the second cell type is mesenchymal stem cells. 
 
     
     
       9. The method of  claim 8 , wherein the injecting is effective to treat a condition of ischemia. 
     
     
       10. The method of  claim 8 , wherein the injecting is performed at multiple locations spaced apart along a limb or other anatomical feature of the patient.